Moleculars and structural studies of the membrane core complex of type IX secretion system

Les maladies parodontales sont causées par une infection bactérienne touchant les tissus entourant les dents, appelés parodonte. L’inflammation aggravée du parodonte peut conduire au déchaussement ou à la chute des dents. Porphyromonas gingivalis est une bactérie anaérobique qui sécrète des toxines appelées gingipaïnes, considérées comme le facteur de virulence majeur. Le système de sécrétion de type IX (T9SS) a été récemment mis en évidence exclusivement chez les membres de la famille des bacteroidetes. Chez P. gingivalis, ce système et directement relié à la sécrétion des gingipaïnes est donc sa pathogénicité. Des études ont montré que plus d’une quinzaine de protéines sont impliquées dans l’assemblage, la fonction et la régulation de ce système de sécrétion. Parmi ces protéines, PorK, PorL, PorM, PorN forment un complexe membranaire au cœur de la machinerie de sécrétion, enchâssé dans les deux membranes bactériennes. L’objectif de ce travail de thèse a été de mettre en place une méthodologie d’extraction et de solubilisation du complexe membranaire PorK-L-M-N afin d’étudier sa structure moléculaire par des méthodes de biochimie intégrative. Trois sous-complexes ont été étudiés successivement. Le complexe de membrane externe PorK-N, le complexe de membrane externe étendu PorK-N-M et le complexe de membrane interne PorL-M. Les résultats obtenus montrent que le complexe de membrane externe PorK-N présente une structure en forme d’anneau de 50nm de diamètre et le complexe de membrane interne PorL-M possède une structure étendue avec une base sphérique de 25nm. Ces résultats valident une méthodologie qui pourra par la suite être utilisée pour d'autres études du T9SS.Periodontal diseases are caused by a bacterial infection affecting the tissues surrounding the teeth, called periodontal. The aggravated inflammation of the periodontium may lead to loosening or falling of the teeth. Porphyromonas gingivalis is an anaerobic bacterium able to secrete toxins called gingipains, considered as the major virulence factor. First called PorSS, the type IX secretion system (T9SS) was recently found exclusively in members of bacteroidetes. In P. gingivalis this system is directly related to the secretion of gingipains and is therefore its pathogenicity. Studies have shown that more than fifteen proteins are involved in the assembly, function and regulation of this secretory system. Among these proteins PorK, PorL, PorM, PorN form a membrane core complex, the central part of the secretory machinery embedded in the two bacterial membranes. The objective of this thesis was to set up a methodology of extraction and solubilization of the PorK-L-M-N membrane complex in order to study its molecular structure by integrative biochemistry methods. Three sub-complexes have been studied successively. PorK-N the outer membrane complex, PorK-N-M extended outer membrane complex and PorL-M the inner membrane complex. The results show that the PorK-N outer membrane complex has a ring-shaped structure of 50nm in diameter, confirming published results, and the PorL-M inner membrane complex has an extended structure of 25nm with a spherical base. These results validate the established methodology that can subsequently be used to continue the structural study of T9SS

Camelid nanobodies used as crystallization chaperones for different constructs of PorM, a component of the type IX secretion system from Porphyromonas gingivalis

International audiencePorM is a membrane protein that is involved in the assembly of the type IX secretion system (T9SS) in Porphyromonas gingivalis, a major bacterial pathogen that is responsible for periodontal disease in humans. In the context of structural studies of PorM to better understand T9SS assembly, four camelid nanobodies were selected, produced and purified, and their specific interaction with the N-terminal or C-terminal part of the periplasmic domain of PorM was investigated. Diffracting crystals were also obtained, and the structures of the four nanobodies were solved by molecular replacement. Furthermore, two nanobodies were used as crystallization chaperones and turned out to be valuable tools in the structure-determination process of the periplasmic domain of PorM

Identification of Dual Receptor Binding Protein Systems in Lactococcal 936 Group Phages

Siphoviridae of the lactococcal 936 group are the most commonly encountered bacteriophages in the dairy processing environment. The 936 group phages possess a discrete baseplate at the tip of their tail—a complex harbouring the Receptor Binding Protein (RBP) which is responsible for host recognition and attachment. The baseplate-encoding region is highly conserved amongst 936 phages, with 112 of 115 publicly available phages exhibiting complete synteny. Here, we detail the three exceptions (Phi4.2, Phi4R15L, and Phi4R16L), which differ from this genomic architecture in possessing an apparent second RBP-encoding gene upstream of the “classical„ rbp gene. The newly identified RBP possesses an elongated neck region relative to currently defined 936 phage RBPs and is genetically distinct from defined 936 group RBPs. Through detailed characterisation of the representative phage Phi4.2 using a wide range of complementary techniques, we demonstrated that the above-mentioned three phages possess a complex and atypical baseplate structure. Furthermore, the presence of both RBPs in the tail tip of the mature virion was confirmed, while the anticipated host-binding capabilities of both proteins were also verified

Strategies for Heterologous Expression, Synthesis, and Purification of Animal Venom Toxins

Animal venoms are complex mixtures containing peptides and proteins known as toxins, which are responsible for the deleterious effect of envenomations. Across the animal Kingdom, toxin diversity is enormous, and the ability to understand the biochemical mechanisms governing toxicity is not only relevant for the development of better envenomation therapies, but also for exploiting toxin bioactivities for therapeutic or biotechnological purposes. Most of toxinology research has relied on obtaining the toxins from crude venoms; however, some toxins are difficult to obtain because the venomous animal is endangered, does not thrive in captivity, produces only a small amount of venom, is difficult to milk, or only produces low amounts of the toxin of interest. Heterologous expression of toxins enables the production of sufficient amounts to unlock the biotechnological potential of these bioactive proteins. Moreover, heterologous expression ensures homogeneity, avoids cross-contamination with other venom components, and circumvents the use of crude venom. Heterologous expression is also not only restricted to natural toxins, but allows for the design of toxins with special properties or can take advantage of the increasing amount of transcriptomics and genomics data, enabling the expression of dormant toxin genes. The main challenge when producing toxins is obtaining properly folded proteins with a correct disulfide pattern that ensures the activity of the toxin of interest. This review presents the strategies that can be used to express toxins in bacteria, yeast, insect cells, or mammalian cells, as well as synthetic approaches that do not involve cells, such as cell-free biosynthesis and peptide synthesis. This is accompanied by an overview of the main advantages and drawbacks of these different systems for producing toxins, as well as a discussion of the biosafety considerations that need to be made when working with highly bioactive proteins

High-Throughput Production of a New Library of Human Single and Tandem PDZ Domains Allows Quantitative PDZ-Peptide Interaction Screening Through High-Throughput Holdup Assay

International audiencePDZ domains recognize PDZ Binding Motifs (PBMs) at the extreme C-terminus of their partner proteins. The human proteome contains 266 identified PDZ domains, the PDZome, spread over 152 proteins. We previously developed the "holdup" chromatographic assay for high-throughput determination of PDZ-PBM affinities. In that work, we had used an expression library of 241 PDZ constructs (the "PDZome V.1"). Here, we cloned, produced, and characterized a new bacterial expression library ("PDZome V.2"), which comprises all the 266 known human PDZ domains as well as 37 PDZ tandem constructs. To ensure the best expression level, folding, and solubility, all construct boundaries were redesigned using available structural data and all DNA sequences were optimized for Escherichia coli expression. Consequently, all the PDZ constructs are produced in a soluble form. Precise quantification and quality control were carried out. The binding profiles previously published using "PDZome V.1" were reproduced and completed using the novel "PDZome V.2" library. We provide here the detailed description of the high-throughput protocols followed through the PDZ gene synthesis and cloning, PDZ production, holdup assay and data treatment

High-throughput production of oxidized animal toxins in Escherichia coli

High-throughput production (HTP) of synthetic genes is becoming an important tool to explore the biological function of the extensive genomic and meta-genomic information currently available from various sources. One such source is animal venom, which contains thousands of novel bioactive peptides with potential uses as novel therapeutics to treat a plethora of diseases as well as in environmentally benign bioinsecticide formulations. Here, we describe a HTP platform for recombinant bacterial production of oxidized disulfide-rich proteins and peptides from animal venoms. High-throughput, host-optimized, gene synthesis and subcloning, combined with robust HTP expression and purification protocols, generate a semiautomated pipeline for the accelerated production of proteins and peptides identified from genomic or transcriptomic libraries. The platform has been applied to the production of thousands of animal venom peptide toxins for the purposes of drug discovery, but has the power to be universally applicable for high-level production of various and diverse target proteins in soluble form. This chapter details the HTP protocol for gene synthesis and production, which supported high levels of peptide expression in the E. coli periplasm using a cleavable DsbC fusion. Finally, target proteins and peptides are purified using automated HTP methods, before undergoing quality control and screening

High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery

International audienceAnimal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane receptors. Disulphide-reticulated peptides have evolved to display improved specificity, low immunogenicity and to show much higher resistance to degradation than linear peptides. These properties make venom peptides attractive candidates for drug development. However, recombinant expression of reticulated peptides containing disulphide bonds is challenging, especially when associated with the production of large libraries of bioactive molecules for drug screening. To date, as an alternative to artificial synthetic chemical libraries, no comprehensive recombinant libraries of natural venom peptides are accessible for high-throughput screening to identify novel therapeutics

An atlas of protein homo-oligomerization across domains of life

Protein structures are essential to understanding cellular processes in molecular detail. While advances in artificial intelligence revealed the tertiary structure of proteins at scale, their quaternary structure remains mostly unknown. We devise a scalable strategy based on AlphaFold2 to predict homo-oligomeric assemblies across four proteomes spanning the tree of life. Our results suggest that approximately 45% of an archaeal proteome and a bacterial proteome and 20% of two eukaryotic proteomes form homomers. Our predictions accurately capture protein homo-oligomerization, recapitulate megadalton complexes, and unveil hundreds of homo-oligomer types, including three confirmed experimentally by structure determination. Integrating these datasets with omics information suggests that a majority of known protein complexes are symmetric. Finally, these datasets provide a structural context for interpreting disease mutations and reveal coiled-coil regions as major enablers of quaternary structure evolution in human. Our strategy is applicable to any organism and provides a comprehensive view of homo-oligomerization in proteomes

The AAA+ ATPase RavA and its binding partner ViaA modulate E. coli aminoglycoside sensitivity through interaction with the inner membrane

International audienceEnteric bacteria have to adapt to environmental stresses in the human gastrointestinal tract such as acid and nutrient stress, oxygen limitation and exposure to antibiotics. Membrane lipid composition has recently emerged as a key factor for stress adaptation. The E. coli ravA-viaA operon is essential for aminoglycoside bactericidal activity under anaerobiosis but its mechanism of action is unclear. Here we characterise the VWA domain-protein ViaA and its interaction with the AAA+ ATPase RavA, and find that both proteins localise at the inner cell membrane. We demonstrate that RavA and ViaA target specific phospholipids and subsequently identify their lipid-binding sites. We further show that mutations abolishing interaction with lipids restore induced changes in cell membrane morphology and lipid composition. Finally we reveal that these mutations render E. coli gentamicin-resistant under fumarate respiration conditions. Our work thus uncovers a ravA-viaA -based pathway which is mobilised in response to aminoglycosides under anaerobiosis and engaged in cell membrane regulation